Rhesus monkey neuropeptide Y Y2 receptor

ABSTRACT

This invention describes a novel rhesus monkey receptor having affinity for neuropeptide Y, pancreatic polypeptide, and peptide YY. This invention also encompasses nucleic acids encoding this receptor, or a fragment thereof, as well as methods employing this receptor and the nucleic acid compounds.

PRIORITY CLAIM

This application claims the benefit of United States Provisional PatentApplication 60/019,809, filed Jun. 17, 1996

BACKGROUND OF THE INVENTION

Neuropeptide Y is a peptide present in the central and peripheralnervous systems. The peptide co-exists with noradrenaline in manyneurons and acts as a neurotransmitter per se or synergisticallytogether with noradrenaline. Neuropeptide Y-containing fibers arenumerous around arteries in the heart, but are also found around thearteries in the respiratory tract, the gastrointestinal tract, and thegenitourinary tract.

Neuropeptide Y is also present in the cerebrum with effects on bloodpressure, feeding, and the release of different hormones. Alterations incentral concentrations of neuropeptide Y have been implicated in theetiology of psychiatric disorders.

Neuropeptide Y was discovered, isolated and sequenced about ten yearsago from porcine brain as part of a general screening protocol todiscover carboxy-terminal amidated peptides and was named neuropeptide Ydue to its isolation form neural tissue and the presence of tyrosine asboth the amino and carboxy terminal amino acid. Neuropeptide Y is amember of the pancreatic family of peptides and shares significantsequence homology with pancreatic polypeptide, and peptide YY.

Neuropeptide Y and the other members of its family of peptides allfeature a tertiary structure consisting of an N-terminal polyprolinehelix and an amphiphilic α-helix, connected with a β-turn, creating ahairpin-like loop, which is sometimes referred to as the pancreaticpolypeptide (PP) fold. The helices are kept together by hydrophobicinteractions. The amidated C-terminal end projects away from the hairpinloop.

Subsequent to its discovery neuropeptide Y was identified as being themost abundant peptide in the central nervous system with widespreaddistribution including the cortex, brainstem, hippocampus, hypothalamus,amygdala, and thalamus as well as being present in the peripheralnervous system in sympathetic neurons and adrenal chromaffin cells.

Neuropeptide Y seems to fulfill the main neurotransmitter criteria,since it is stored in synaptic granules, is released upon electricalnerve stimulation, and acts at specific receptors. It is clear thatneuropeptide Y is an important messenger in its own right, probably inthe brain, where neuropeptide Y potently inhibits the activity ofadenylate cyclase and induces an increase in the intracellular levels ofcalcium. Central injection of neuropeptide Y results in blood pressurechanges, increased feeding, increased fat storage, elevated blood sugarand insulin, decreased locomotor activity, reduced body temperature, andcatalepsy.

Neuropeptide Y (as well as its chemical relatives) acts upon membranereceptors that are dependent on guanine nucleotides, known as Gprotein-coupled receptors. G proteins are a family of membrane proteinsthat become activated only after binding guanosine triphosphate.Activated G proteins in turn activate an amplifier enzyme on the innerface of a membrane; the enzyme then converts precursor molecules intosecond messengers.

Neuropeptide Y appears to interact with a family of closely relatedreceptors. These receptors are generally classified into severalsubtypes based upon the ability of different tissues and receptors tobind different fragments of neuropeptide Y and the closely relatedpeptide YY.

The Y1 receptor subtype appears to be the major vascular neuropeptide Yreceptor. The Y2 receptor subtypes can also occur postjunctionally onvascular smooth muscle. The as-yet-unisolated Y3 receptor subtypeappears to be neuropeptide Y-specific, not binding peptide YY. Thisreceptor is likely to be present in the adrenal tissues, medulla, heart,and brain stem, among other areas. [For a review of neuropeptide Y andneuropeptide Y receptors, see. e.g., C. Wahlestedt and D. Reis, AnnualReview of Pharmacology and Toxicolog, 33:309-352 (1993)].

In view of the wide number of clinical maladies associated with anexcess of neuropeptide Y and related peptides, the development ofneuropeptide Y receptor antagonists will serve to control these clinicalconditions. The earliest such receptor antagonists were peptidederivatives. These antagonists proved to be of limited pharmaceuticalutility because of their metabolic instability.

The present invention provides an additional receptor from the rehesusmonkey neuropeptide Y receptor family, the receptor of the presentinvention being designated the Y2 receptor, to those previously known.The characterization and treatment of physiological disorders is herebyfurthered.

SUMMARY OF THE INVENTION

This invention provides an isolated amino acid compound useful as areceptor for neuropeptide Y and related peptides, said compoundcomprising the amino acid sequence

Met Gly Pro Ile Gly Thr Glu Ala Asp Glu Asn Gln Thr Val Glu Glu  1               5                  l0                  l5 Met Lys ValGlu Gln Tyr Gly Pro Gln Thr Thr Pro Arg Gly Glu Leu             20                  25                  30 Val Pro Asp ProGlu Pro Glu Leu Ile Asp Ser Thr Lys Leu Ile Glu         35                  40                  45 Val Gln Val Val LeuIle Leu Ala Tyr Cys Ser Ile Ile Leu Leu Gly     50                  55                  60 Val Ile Gly Asn Ser LeuVal Ile His Val Val Ile Lys Phe Lys Ser 65                  70                  75                  80 Met ArgThr Val Thr Asn Phe Phe Ile Ala Asn Leu Ala Val Ala Asp                 85                  90                  95 Leu Val ValAsn Thr Leu Cys Leu Pro Phe Thr Leu Thr Tyr Thr Leu            100                 105                 110 Met Gly Glu TrpLys Met Gly Pro Val Leu Cys Hls Leu Val Pro Tyr        115                 120                 125 Ala Gln Gly Leu AlaVal Gln Val Ser Thr Ile Thr Leu Thr Val Ile    130                 135                 140 Ala Leu Asp Arg His ArgCys Ile Val Tyr His Leu Glu Ser Lys Ile145                 150                 155                 160 Ser LysArg Ile Ser Phe Leu Ile Ile Gly Leu Ala Trp Gly Ile Ser                165                 170                 175 Ala Leu LeuAla Ser Pro Leu Ala Ile Phe Arg Glu Tyr Ser Leu Ile            180                 185                 190 Glu Ile Ile ProAsp Phe Glu Ile Val Ala Cys Thr Glu Lys Trp Pro        195                 200                 205 Gly Glu Glu Lys SerIle Tyr Gly Thr Val Tyr Ser Leu Ser Ser Leu    210                 215                 220 Leu Ile Leu Tyr Val LeuPro Leu Gly Ile Ile Ser Phe Ser Tyr Thr225                 230                 235                 240 Arg IleTrp Ser Lys Leu Lys Ser His Val Ser Pro Gly Ala Ala Asn                245                 250                 255 Asp His TyrHis Gln Arg Arg Gln Lys Thr Thr Lys Met Leu Val Cys            260                 265                 270 Val Val Val Va1Phe Ala Val Ser Trp Leu Pro Leu His Ala Phe Gln        275                 280                 285 Leu Ala Val Asp IleAsp Ser His Val Leu Asp Leu Lys Glu Tyr Lys    290                 295                 300 Leu Ile Phe Thr Val PheHis Ile Ile Ala Met Cys Ser Thr Phe Ala305                 310                 315                 320 Asn ProLeu Leu Tyr Gly Trp Met Asn Ser Asn Tyr Arg Lys Ala Phe                325                 330                 335 Leu Ser AlaPhe Arg Cys Glu Gln Arg Leu Asp Ala Ile His Ser Glu            340                 345                 350 Val Ser Val ThrPhe Lys Ala Lys Lys Asn Leu Glu Val Arg Lys Asn        355                 360                 365 Ser Gly Pro Asn AspSer Phe Thr Glu Ala Thr Asn Val    370                 375                 380

hereinafter designated as SEQ ID NO:2.

The invention also provides an isolated nucleic acid compound thatcomprises a nucleic acid sequence which encodes the amino acid compoundsprovided. Particularly this invention provides the isolated nucleic acidcompound having the sequence

TCATTGAGGT ACAAGTTGTA GACTCTTGTG CTGGTTGCAC GCCAAGTGGA ACTGTACTGA 60 AAATG GGT CCA ATA GGT ACA GAG CCT GAT GAG AAC CAG ACA GTG GAA 107    MetGly Pro Ile Gly Thr Glu Ala Asp Glu Asn Gln Thr Val Glu     1               5                  10                  15 GAA ATGAAG GTG GAA CAA TAT GGG CCA CAA ACC ACT CCT AGA GGT GAA 155 Glu Met LysVal Glu Gln Tyr Gly Pro Gln Thr Thr Pro Arg Gly Glu                 20                  25                  30 CTG GTC CCTGAT CCT GAG CCA GAG CTT ATA GAT AGT ACC AAG CTG ATT 203 Leu Val Pro AspPro Glu Pro Glu Leu Ile Asp Ser Thr Lys Leu Ile             35                  40                  45 GAG GTA CAA GTTGTC CTC ATA TTG GCC TAT TGC TCC ATC ATC TTG CTT 251 Glu Val Gln Val ValLeu Ile Leu Ala Tyr Cys Ser Ile Ile Leu Leu         50                  55                  60 GGG GTA ATT GGC AACTCC TTG GTG ATC CAC GTG GTG ATC AAA TTC AAG 299 Gly Val Ile Gly Asn SerLeu Val Ile His Val Val Ile Lys Phe Lys     65                  70                  75 AGC ATG CGC ACA GTA ACCAAC TTT TTC ATC GCC AAT CTG GCT GTG GCA 347 Ser Met Arg Thr Val Thr AsnPhe Phe Ile Ala Asn Leu Ala Val Ala 80                  85                  90                  95 GAT CTTGTG GTG AAT ACT CTG TGT CTA CCA TTC ACT CTT ACC TAC ACC 395 Asp Leu ValVal Asn Thr Leu Cys Leu Pro Phe Thr Leu Thr Tyr Thr                100                 105                 110 TTA ATG GGGGAG TGG AAA ATG GGT CCT GTC CTG TGC CAC CTG GTG CCC 443 Leu Met Gly GluTrp Lys Met Gly Pro Val Leu Cys His Leu Val Pro            115                 120                 125 TAT GCA CAG GGCCTG GCA GTA CAA GTA TCC ACA ATC ACC TTG ACA GTA 491 Tyr Ala Gln Gly LeuAla Val Gln Val Ser Thr Ile Thr Leu Thr Val        130                 135                 140 ATT GCC CTG GAC CGGCAC AGG TGC ATC GTC TAC CAC CTG GAG AGC AAG 539 Ile Ala Leu Asp Arg HisArg Cys Ile Val Tyr His Leu Glu Ser Lys    145                 150                 155 ATC TCC AAG CGT ATC AGCTTC CTG ATT ATT GGC TTG GCC TGG GGC ATC 587 Ile Ser Lys Arg Ile Ser PheLeu Ile Ile Gly Leu Ala Trp Gly Ile160                 165                 170                 175 AGT GCCCTG CTA GCA AGT CCC CTG GCC ATC TTC CGG GAG TAT TCA CTG 635 Ser Ala LeuLeu Ala Ser Pro Leu Ala Ile Phe Arg Glu Tyr Ser Leu                180                 185                 190 ATT GAG ATCATT CCG GAT TTT GAG ATT GTG GCC TGT ACT GAA AAA TGG 683 Ile Glu Ile IlePro Asp Phe Glu Ile Val Ala Cys Thr Glu Lys Trp            195                 200                 205 CCT GGC GAG GAAAAG AGC ATC TAT GGC ACT GTC TAC AGT CTT TCT TCC 731 Pro Gly Glu Glu LysSer Ile Tyr Gly Thr Val Tyr Ser Leu Ser Ser        210                 215                 220 TTG TTG ATC CTG TACGTT TTG CCT CTG GGC ATA ATA TCA TTT TCC TAC 779 Leu Leu Ile Leu Tyr ValLeu Pro Leu Gly Ile Ile Ser Phe Ser Tyr    225                 230                 235 ACT CGC ATT TGG AGT AAATTG AAG AGC CAT GTC AGT CCT GGA GCT GCA 827 Thr Arg Ile Trp Ser Lys LeuLys Ser His Val Ser Pro Gly Ala Ala240                 245                 250                 255 AAT GACCAC TAC CAT CAG CGA AGG CAA AAA ACC ACC AAA ATG CTG GTG 875 Asn Asp HisTyr His Gln Arg Arg Gln Lys Thr Thr Lys Met Leu Val                260                 265                 270 TGC GTG GTGGTG GTG TTT GCG GTC AGC TGG CTG CCT CTC CAT GCC TTC 923 Cys Val Val ValVal Phe Ala Val Ser Trp Leu Pro Leu His Ala Phe            275                 280                 285 CAG CTT GCC GTTGAC ATT GAC AGC CAT GTC CTG GAC CTG AAG GAG TAC 971 Gln Leu Ala Val AspIle Asp Ser His Val Leu Asp Leu Lys Glu Tyr        290                 295                 300 AAA CTC ATC TTC ACAGTG TTC CAC ATC ATC GCC ATG TGC TCC ACT TTT 1019 Lys Leu Ile Phe Thr ValPhe His Ile Ile Ala Met Cys Ser Thr Phe    305                 310                 315 GCC AAT CCC CTT CTC TATGGC TGG ATG AAC AGC AAC TAT AGA AAG GCT 1067 Ala Asn Pro Leu Leu Tyr GlyTrp Met Asn Ser Asn Tyr Arg Lys Ala320                 325                 330                 335 TTC CTCTCT GCC TTC CGC TGT GAG CAG CGG TTG GAT GCC ATT CAC TCT 1115 Phe Leu SerAla Phe Arg Cys Glu Gln Arg Leu Asp Ala Ile His Ser                340                 345                 350 GAG GTG TCCGTG ACA TTC AAG GCT AAA AAG AAC CTG GAG GTC AGA AAA 1163 Glu Val Ser ValThr Phe Lys Ala Lys Lys Asn Leu Glu Val Arg Lys            355                 360                 365 AAT AGT GGC CCCAAT GAC TCT TTC ACA GAA GCT ACC AAT GTC 1205 Asn Ser Gly Pro Asn Asp SerPhe Thr Glu Ala Thr Asn Val        370                 375                 380 TAAGGAAGCTAGGGTGTGAA AATGTATGAA TGAATTCTGA CCAGAGCTAT AAATCTGGTT 1265 GATGGCGGCTCACAAGTGAT AATTGATTTC CCATTTTAAG GAAGAAGAGG ATCTAAATGG 1325 AAGCATCTGCTGTTTAGTTC CTGGAAAACT GGCTGGGAAG AGCCTGTGTG AAAATACTTG 1385 AATTCAAAGATAAGGCAGCA AAATGGTTTA CTTAACAGTT GGTAGGGTAG TAGGTTGAAT 1445 TAGGAGTAAAAGCAGAGAGA GGTACTTTTG ACTATTTTCC TGGAGTGAAG TAAACTTGAA 1505 CAAGGAATTGGTATTATCAG CATTGCAAAG AGACGGTGGG TAAATAAGTT GATTTTCAGA 1565 TTTCATTAGGACCTGGATTG GGGAGCTGTG TAGTTCACGG TTCCCTGCTT GGCTGATGAA 1625 AACGTCGCTGAACAAAAATT TCTCCAGGGA GCCACAGGCT CTCCTTCATC ACGTTTTGAT 1685 TTTTTTTGTTAATTCTCTAG ACAAAATCCA TCAAGGAATG CTGCAGGAAA AGATTGCCAG 1745 CTATATGAATGGCTTCAAGG AACTAAACTG AAACTTGCTA TATAATTAAT ATTTTGGCAG 1805 ACGATAGGGGAACTCCTCAA CACTCAGTGA GCCAATTGTT CTTAAAACCG GTTGCACATT 1865 TGGTGAAAGTTTCTTCAACT CTGAATCAAA AGCTGAAATT CTCAGAATTG CAGGAAATGC 1925 AAACCATCATTTAATTTGTA ATTTCAAGTT ACATCTGCTT TATGGAGATA TTTAGATAAC 1985 AAGCATACAACTTGATAGTT TTATTGTTAT ACCTTTTTGA ACATGTATGA TTTATGTTAT 2045 TATTCCTATTGGAGCTAAGT TTGTCTACAC TAAAATTTAA ATCAGAATAA AGAATAATTT 2105 TTGTGGAAAAAAAAAAAAAA AAAAAAAAAA AAACTCGAG 2144

which is hereinafter designated as SEQ ID NO:1.

This invention also provides recombinant nucleic acid vectors comprisingnucleic acids encoding SEQ ID NO:2. This invention also encompassesrecombinant DNA vectors which comprise the isolated DNA sequence whichis SEQ ID NO:1.

The present invention also provides assays for determining the efficacyand adverse reaction profile of agents useful in the treatment orprevention of disorders associated with an excess or deficiency in theamount of neuropeptide Y present.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

The terms and abbreviations used in this document have their normalmeanings unless otherwise designated. For example “C” refers to degreesCelsius; “N” refers to normal or normality; “mmol” refers to millimoleor millimoles; “g” refers to gram or grams; “ml” means milliter ormilliters; “M” refers to molar or molarity; “μg” refers to microgram ormicrograms; and “μl” refers to microliter or microliters.

All nucleic acid sequences, unless otherwise designated, are written inthe direction from the 5′ end to the 3′ end, frequently referred to as“5′ to 3′”.

All amino acid or protein sequences, unless otherwise designated, arewritten commencing with the amino terminus (“N-terminus”) and concludingwith the carboxy terminus (“C-terminus”). “Base pair” or “bp” as usedherein refers to DNA or RNA. The abbreviations A,C,G, and T correspondto the 5′-monophosphate forms of the deoxyribonucleosides(deoxy)adenine, (deoxy)cytidine, (deoxy)guanine, and (deoxy)thymine,respectively, when they occur in DNA molecules. The abbreviations U,C,G,and T correspond to the 5′-monophosphate forms of the ribonucleosidesuracil, cytidine, guanine, and thymine, respectively when they occur inRNA molecules. In double stranded DNA, base pair may refer to apartnership of A with T or C with G. In a DNA/RNA, heteroduplex basepair may refer to a partnership of A with U or C with G. (See thedefinition of “complementary”, infra.)

The terms “digestion” or “restriction” of DNA refers to the catalyticcleavage of the DNA with a restriction enzyme that acts only at certainsequences in the DNA (“sequence-specific endonucleases”). The variousrestriction enzymes used herein are commercially available and theirreaction conditions, cofactors, and other requirements were used aswould be known to one of ordinary skill in the art. Appropriate buffersand substrate amounts for particular restriction enzymes are specifiedby the manufacturer or can be readily found in the literature.

“ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments. Unless otherwise provided,ligation may be accomplished using known buffers and conditions with aDNA ligase, such as T4 DNA ligase.

The term “plasmid” refers to an extrachromosomal (usually)self-replicating genetic element. Plasmids are generally designated by alower case “p” preceded and/or followed by letters and/or numbers. Thestarting plasmids herein are either commercially available, publiclyavailable on an unrestricted basis, or can be constructed from availableplasmids in accordance with published procedures. In addition,equivalent plasmids to those described are known in the art and will beapparent to the ordinarily skilled artisan.

The term “reading frame” means the nucleotide sequence from whichtranslation occurs “read” in triplets by the translational apparatus oftransfer RNA (tRNA) and ribosomes and associated factors, each tripletcorresponding to a particular amino acid. A base pair insertion ordeletion (termed a frameshift mutation) may result in two differentproteins being coded for by the same DNA segment. To insure againstthis, the triplet codons corresponding to the desired polypeptide mustbe aligned in multiples of three from the initiation codon, i.e. thecorrect “reading frame” being maintained.

“Recombinant DNA cloning vector” as used herein refers to anyautonomously replicating agent, including, but not limited to, plasmidsand phages, comprising a DNA molecule to which one or more additionalDNA segments can or have been added.

The term “recombinant DNA expression vector” as used herein refers toany recombinant DNA cloning vector in which a promoter to controltranscription of the inserted DNA has been incorporated.

The term “expression vector system” as used herein refers to arecombinant DNA expression vector in combination with one or moretrans-acting factors that specifically influence transcription,stability, or replication of the recombinant DNA expression vector. Thetrans-acting factor may be expressed from a co-transfected plasmid,virus, or other extrachromosomal element, or may be expressed from agene integrated within the chromosome.

“Transcription” as used herein refers to the process whereby informationcontained in a nucleotide sequence of DNA is transferred to acomplementary RNA sequence.

The term “transfection” as used herein refers to the taking up of anexpression vector by a host cell whether or not any coding sequences arein fact expressed. Numerous methods of transfection are known to theordinarily skilled artisan, for example, calcium phosphateco-precipitation, and electroporation. Successful transfection isgenerally recognized when any indication of the operation of this vectoroccurs within the host cell.

The term “transformation” as used herein means the introduction of DNAinto an organism so that the DNA is replicable, either as anextrachromosomal element or by chromosomal integration. Methods oftransforming bacterial and eukaryotic hosts are well known in the art,many of which methods, such as nuclear injection, protoplast fusion orby calcium treatment using calcium chloride are summarized in J.Sambrook, etal, MOLECULAR CLONING: A LABORATORY MANUAL, (1989).

The term “translation” as used herein refers to the process whereby thegenetic information of messenger RNA is used to specify and direct thesynthesis of a polypeptide chain.

The term “vector” as used herein refers to a nucleic acid compound usedfor the transformation of cells in gene manipulation bearingpolynucleotide sequences corresponding to appropriate protein moleculeswhich when combined with appropriate control sequences confer specificproperties on the host cell to be transformed. Plasmids, viruses, andbacteriophage are suitable vectors. Artificial vectors are constructedby cutting and joining DNA molecules from different sources usingrestriction enzymes and ligases. The term “vector” as used hereinincludes Recombinant DNA cloning vectors and Recombinant DNA expressionvectors.

The terms “complementary” or “complementarity” as used herein refers topair of bases, purines and pyrimidines, that associate through hydrogenbonding in double stranded nucleic acid. The following base pairs arecomplementary: guanine and cytosine; adenine and thymine; and adenineand uracil.

The term “hybridization” as used herein refers to a process in which astrand of nucleic acid joins with a complementary strand through basepairing. The conditions employed in the hybridization of twonon-identical, but very similar, complementary nucleic acids varies withthe degree of complementarity of the two strands and the length of thestrands. Such techniques and conditions are well known to practitionersin this field.

“Isolated amino acid sequence” refers to any amino acid sequence,however constructed or synthesized, which is locationally distinct fromthe naturally occurring sequence.

“Isolated DNA compound” refers to any DNA sequence, however constructedor synthesized, which is locationally distinct from its natural locationin genomic DNA.

“Isolated nucleic acid compound” refers to any RNA or DNA sequence,however constructed or synthesized, which is locationally distinct fromits natural location.

A “primer” is a nucleic acid fragment which functions as an initiatingsubstrate for enzymatic or synthetic elongation.

The term “promoter” refers to a DNA sequence which directs transcriptionof DNA to RNA.

A “probe” as used herein is a nucleic acid compound or a fragmentthereof which hybridizes with a nucleic acid compound which encodeseither the entire sequence SEQ ID NO:2, a sequence complementary to SEQID NO:2, or a part thereof.

The term “stringency” refers to a set of hybridization conditions whichmay be varied in order to vary the degree of nucleic acid affinity forother nucleic acid. (See the definition of “hybridization”, supra.)

The term “antigenically distinct” as used herein refers to a situationin which antibodies raised against an epitope of the proteins of thepresent invention, or a fragment thereof, may be used to differentiatebetween the proteins of the present invention and other neuropeptide Yreceptor subtypes. This term may also be employed in the sense that suchantibodies may be used to differentiate between the rhesus monkey Y2receptor protein and analogous proteins derived from other species.

The term “PCR” as used herein refers to the widely-known polymerasechain reaction employing a thermally-stable polymerase.

This invention provides the protein of SEQ ID NO:2, a rhesus monkeyneuropeptide Y receptor, designated as a Y2 receptor. [For a review ofneuropeptide Y receptors, see, D. Gehlert, Life Sciences, 55:551-562(1994)]. Traditional receptors of this family have considerable overlapin their binding affinities for neuropeptide Y and peptide YY whilepancreatic polypeptide appears to have its own distinct set ofreceptors. Many, but not all, of the effects of neuropeptide Y can bereplicated using peptide YY. The receptor of the present invention, asdescribed infra, has considerable pharmacological overlap betweenpancreatic polypeptide and peptide YY and less affinity for neuropeptideY, indicating it belongs to a novel subclass of receptors.

Two subtypes of receptors for neuropeptide Y were initially proposed onthe basis of the affinity of the 13-36 fragment of neuropeptide Y usinga preparation of the sympathetic nervous system. While these are thebest established receptors for neuropeptide Y, a substantial body ofevidence exists that there are additional receptor subtypes. The bestestablished is a Y-3 receptor that is responsive to neuropeptide Y, butnot to peptide YY. Another recently delineated receptor has beendescribed that binds peptide YY with high affinity and neuropeptide Ywith lower affinity. While the pharmacology of the feeding response toneuropeptide Y appears to be Y-1 in nature, a separate “feedingreceptor” has been proposed. Until this invention, the Y-1 receptor wasthe only one that had been successfully cloned to date. The followingparagraphs summarize the available information on the known neuropeptideY receptor subtypes and their potential role in physiological function.

Y-1 Receptor

The Y-1 receptor is the best characterized receptor for neuropeptide Y.This receptor is generally considered to be postsynaptic and mediatesmany of the known actions of neuropeptide Y in the periphery.Originally, this receptor was described as having poor affinity forC-terminal fragments of neuropeptide Y, such as the 13-36 fragment, butinteracts with the full length neuropeptide Y and peptide YY with equalaffinity. L. Selbie, etal., Patent Cooperation Treaty publication WO93/09227, published May 13, 1993; C. Wahlestedt, etal., RegulatoryPotides, 13:307-318 (1986); C. Wahlestedt, etal., NEURONAL MESSENGERS INVASCULAR FUNCTION, 231-241 (Nobin, etal., eds. 1987). Substitution ofthe amino acid at position 34 with a proline (Pro³⁴) results in aprotein which is specific for the Y-1 receptor. E. K. Potter, etal.,European Journal of Pharmacology, 193:15-19 (1991). This tool has beenused to establish a role for the Y-1 receptor in a variety of functions.The receptor is thought to be coupled to adenylate cyclase in aninhibitory manner in cerebral cortex, vascular smooth muscle cells, andSK-N-MC. [For a review, see, B.J. McDermott, et al, CardiovascularResearch, 27:893-905 (1993)]. This action is prevented by application ofpertussis toxin confirming the role of a G-protein coupled receptor. TheY-1 receptor mediates the mobilization of intracellular calcium in aporcine vascular smooth muscle cells and human erythroleukemia cells.

The cloned human Y-1 receptor can couple to either phosphotidylinositolhydrolysis or the inhibition of adenylate cyclase, depending on the typeof cell in which the receptor is expressed. H. Herzog, e al, Proceedingsof the National Academy of Sciences (USA), 89:5794-5798 (1992). The Y-1receptor has been reported to couple to either second messenger systemwhen studied using tissue preparations or cell lines naturallyexpressing the receptor. D. Gehlert, supra, at 553. The Y-1 receptorcannot, therefore, be distinguished solely on the basis of coupling to asingle second messenger.

Y-2 Receptor

As with the Y-1 receptor, this receptor subtype was first delineatedusing vascular preparations. Pharmacologically, the Y-2 receptor isdistinguished from Y-1 by exhibiting affinity for C-terminal fragmentsof neuropeptide Y. The receptor is most often differentiated by the useof neuropeptide Y(13-36), though the 3-36 fragment of neuropeptide Y andpeptide YY provides improved affinity and selectivity. Y. Dumont, etal., Society for Neuroscience Abstracts, 19:726 (1993). like Y-1receptor, this receptor is coupled to the inhibition of adenylatecyclase, though in some preparations it may not be sensitive topertussis toxin. The Y-2 receptor was found to reduce the intracellularlevels of clacium in the synspse by selective inhibition of N-typecalcium channels. Like the Y-1 receptor, the Y-2 receptor may exhibitdifferential coupling to second messengers.

The Y-2 receptors are found in a variety of brain regions, including thehippocampus, substantia nigra-lateralis, thalamus, hypothalamus, andbrainstem. In the periphery, Y-2 is found in the peripheral nervoussystem, such as sympathetic, parasympathetic, and sensory neurons. Inall these tissues, Y-2 receptors mediate a decrease in the release ofneurotransmitters.

Y-3 Receptor

This receptor is the newest and least studied of the establishedneuropeptide Y receptor subtypes. While neuropeptide Y is a fullyefficacious agonist at this receptor population, peptide YY is weaklyefficacious. This pharmacological property is used to define thisreceptor. A receptor that has similar pharmacology to the Y-3 receptorhas been identified in the CA3 region of the hippocampus usingelectrophysiological techniques. This receptor may potentiate theexcitatory response of these neurons to N-methyl-D-aspartate (NMIA). F.P. Monnet, etal, European Journal of Pharmacology 35 182:207-208 (1990).

The presence of this receptor is best established in the rhesus monkeybrainstem, specifically in the nucleus tractus solitarius. Applicationof neuropeptide Y to this region produces a dose-dependent reduction inblood pressure and heart rate. This area of the brain also may havesignificant contributions from the Y-1 and Y-2 receptor. Neuropeptide Yalso inhibits the acetylcholine-induced release of catecholamines fromthe adrenal medulla, presumably through a Y-3 receptor. C. Wahlestedt,et al, Life Scenes, 50:PL7-PL14 (1992).

Peptide YY Preferring Receptor

A fourth receptor has been described that exhibits a modest preferencefor peptide YY over neuropeptide Y. This receptor was first described inthe rhesus monkey small intestine as having a 5-10 fold higher affinityfor peptide YY over neuropeptide Y. M. Laburthe, et al., Life Endocrin,118:1910-1917 (1986). Subsequently, this receptor was found in theadipocyte and a kidney proximal tubule cell line. This receptor iscoupled in an inhibitory manner to adenylate cyclase and is sensitive topertussis toxin.

In the intestine, this receptor produces a potent inhibition of fluidand electrolyte secretion. The receptor is localized to the crypt cellswhere intestinal chloride secretion is believed to take place. Thepeptide YY preferring receptor in adipocytes mediates a reduction inlipolysis by way of a cyclic adenosine monophosphate (cAMP)-dependentmechanism.

“Feeding Receptor”

One of the earliest discovered central effects of neuropeptide Y was aprofound increase in food intake that was observed following thehypothalmic administration of the peptide to rats. The response wasgreatest when the peptide was infused into the perifornical region ofthe hypothalamus. B. G. Stanley, et al., Brain Research, 604:304-317(1993). While the pharmacology of this response resembled the Y-1receptor, the 2-36 fragment of neuropeptide Y was significantly morepotent than neuropeptide Y. In addition, intracerebroventricularneuropeptide Y(2-36) fully stimulates feeding, but does not reduce bodytemperature as does full length neuropeptide Y. F. B. Jolicoeur, et al.,Brain Research Bulletin, 26:309-311 (1991).

The receptors of the present invention are believed to potentiatecentral nervous system responses and is, therefore, an important targetfor pharmaceutical purposes. The receptor of the present invention willbe useful in identifying compounds useful in the treatment or preventionof conditions associated with an excess of neuropeptide Y. The term“physiological disorder associated with an inappropriate amount ofneuropeptide Y, peptide YY, or pancreatic polypeptide” encompasses thosedisorders associated with an inappropriate stimulation of a receptor ofthese neuropeptides, regardless of the actual amount of the neuropeptidepresent in the locale.

These physiological disorders include:

disorders or diseases pertaining to the heart, blood vessels or therenal system, such as vasospasm, heart failure, shock, cardiachypertrophy, increased blood pressure, angina, myocardial infarction,sudden cardiac death, arrythmia, peripheral vascular disease, andabnormal renal conditions such as impaired flow of fluid, abnormal masstransport, or renal failure;

conditions related to increased sympathetic nerve activity for example,during or after coronary artery surgery, and operations and surgery inthe gastrointestinal tract;

cerebral diseases and diseases related to the central nervous system,such as cerebral infarction, neurodegeneration, epilepsy, stroke, andconditions related to stroke, cerebral vasospasm and hemorrhage,depression, anxiety, schizophrenia, and dementia;

conditions related to pain or nociception;

diseases related to abnormal gastrointestinal motility and secretion,such as different forms of ileus, urinary incontinence, and Crohn'sdisease;

abnormal drink and food intake disorders, such as obesity, anorexia,bulimia, and metabolic disorders;

diseases related to sexual dysfunction and reproductive disorders;

conditions or disorders associated with inflammation;

respiratory diseases, such as asthma and conditions related to asthmaand bronchoconstriction; and

diseases related to abnormal hormone release, such as leutinizinghormone, growth hormone, insulin, and prolactin.

Skilled artisans will recognize that the proteins of the presentinvention can be synthesized by a number of different methods. All ofthe amino acid compounds of the invention can be made by chemicalmethods well known in the art, including solid phase peptide synthesis,or recombinant methods. Both methods are described in U.S. Pat. No.4,617,149, herein incorporated by reference.

The principles of solid phase chemical synthesis of polypeptides arewell known in the art and may be found in general texts in the area. Seee.g., H. Dugas and C. Penney, BIOORGANIC CHEMSTRY, (1981)Springer-Verlag, New York, pgs. 54-92. For examples, peptides may besynthesized by solid-phase methodology utilizing an Applied Biosystems430A peptide synthesizer (commercially available from AppliedBiosystems, Foster City Calif.) and synthesis cycles supplied by AppliedBiosystems. Protected amino acids, such as t-butoxycarbonyl-protectedamino acids, and other reagents are commercially available from manychemical supply houses.

Sequential t-butoxycarbonyl chemistry using double couple protocols areapplied to the starting-methyl benzhydryl amine resins for theproduction of C-terminal carboxamides. For the production of C-terminalacids, the corresponding pyridine-2-aldoxime methiodide resin is used.Asparagine, glutamine, and arginine are coupled using preformed hydroxybenzotriazole esters. The following side chain protection may be used:

Arg, Tosyl

Asp, cyclohexyl

Glu, cyclohexyl

Ser, Benzyl

Thr, Benzyl

Tyr, 4-bromo carbobenzoxy

Removal of the t-butoxycarbonyl moiety (deprotection) may beaccomplished with trifluoroacetic acid (TFA) in methylene chloride.Following completion of the synthesis the peptides may be deprotectedand cleaved from the resin with anhydrous hydrogen fluoride containing10% meta-cresol. Cleavage of the side chain protecting group(s) and ofthe peptide from the resin is carried out at zero degrees centigrade orbelow, preferably—20_C for thirty minutes followed by thirty minutes atO_C.

After removal of the hydrogen fluoride, the peptide/resin is washed withether, and the peptide extracted with glacial acetic acid and thenlyophilized. Purification is accomplished by size-exclusionchromatography on a Sephadex G-10 (Pharmacia) column in 10% acetic acid.

The proteins of the present invention may also be produced byrecombinant methods. Recombinant methods are preferred if a high yieldis desired. A general method for the construction of any desired DNAsequence is provided in J. Brown, et al, Methods in Enzymology, 68:109(1979). See also, J. Sambrook, et al., supra.

The basic steps in the recombinant production of desired proteins are:

a) construction of a synthetic or semi-synthetic DNA encoding theprotein of interest;

b) integrating said DNA into an expression vector in a manner suitablefor the expression of the protein of interest, either alone or as afusion protein;

c) transforming an appropriate eukaryotic or prokaryotic host cell withsaid expression vector,

d) culturing said transformed or transfected host cell in a manner toexpress the protein of interest; and

e) recovering and purifying the recombinantly produced protein ofinterest.

In general, prokaryotes are used for cloning of DNA sequences inconstructing the vectors of this invention. Prokaryotes may also beemployed in the production of the protein of interest. For example, theEscherichia coli K12 strain 294 (ATCC No. 31446) is particularly usefulfor the prokaryotic expression of foreign proteins. Other strains of E.coli which may be used (and their relevant genotypes) include thefollowing.

Strain Genotype DH5α F⁻ (φ80dlacZΔM15), Δ(lacZYA-argF)U169 supE44, λ⁻,hsdR17(r_(K) ⁻, m_(K) ⁺), recA1, endA1, gyrA96, thi-1, relA1 HB101supE44, hsdS20(r_(B) ⁻m_(B) ⁻), recA13, ara-14, proA₂ lacY1, galK2,rpsL20, xyl-5, mtl-1, mcrB, mrr JM109 recA1, e14⁻(mcrA), supE44, endA1,hsdR17(r_(K) ⁻, m_(k) ⁺), gyrA96, relA1, thi-1,   (lac-proAB),F′[traD36, proAB+ lacI^(q),lacZ   M15] RRI supE44, hsdS20(r_(B) ⁻m_(B)⁻), ara-14 proA₂, lacY1, galK2, rpsL20, xyl-5, mtl-5 χ1776 F⁻, ton, A53,dapD8, minA1, supE42 (glnV42), Δ(gal-uvrB)40, minB2, rfb-2, gyrA₂5,thyA142, oms-2, metC65, oms-1, Δ(bioH-asd)29, cycB2, cycA1, hsdR2, λ⁻294 endA, thi⁻, hsr⁻, hsm_(k) ⁺ (U.S. Pat. No. 4,366,246) LE392 F⁻,hsdR514 (r⁻m⁻), supE44, supF58, lacY1, or Δlac(I-Y)6, galK2, glaT22,metB1, trpR55, λ⁻

These strains are all commercially available from suppliers such as:Bethesda Research Laboratories, Gaithersburg, Md. 20877 and StratageneCloning Systems, La Jolla, Calif. 92037; or are readily available to thepublic from sources such as the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md., 10852-1776.

Except where otherwise noted, these bacterial strains can be usedinterchangeably. The genotypes listed are illustrative of many of thedesired characteristics for choosing a bacterial host and are not meantto limit the invention in any way. The genotype designations are inaccordance with standard nomenclature. See, for example, J. Sambrook, etal., supra. A preferred strain of E. coli employed in the cloning andexpression of the genes of this invention is RV308, which is availablefrom the ATCC under accession number ATCC 31608, and is described inU.S. Pat. No. 4,551,433, issued Nov. 5, 1985.

In addition to the strains of E. coli discussed supra, bacilli such asBacillus subtilis, other enterobacteriaceae such as Salmonellatyphimurium or Serratia marcescans, and various Pseudomonas species maybe used. In addition to these gram-negative bacteria, other bacteria,especially Streptomyces, spp., may be employed in the prokaryoticcloning and expression of the proteins of this invention.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase [vector pGX2907 (ATCC 39344) contains the replicon andβ-lactamase gene] and lactose promoter systems [Chang et al., Nature(London), 275:615 (1978); and Goeddel et al., Nature (London, 281:544(1979)], alkaline phosphatase, the tryptophan (trp) promoter system[vector pATH1 (ATCC 37695) is designed to facilitate expression of anopen reading frame as a trpE fusion protein under control of the trppromoter] and hybrid promoters such as the tac promoter (isolatable fromplasmid pDR540 ATCC-37282). However, other functional bacterialpromoters, whose nucleotide sequences are generally known, enable one ofskill in the art to ligate them to DNA encoding the proteins of theinstant invention using linkers or adapters to supply any requiredrestriction sites. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno sequence operably linked to the DNA encodingthe desired polypeptides. These examples are illustrative rather thanlimiting.

The proteins of this invention may be synthesized either by directexpression or as a fusion protein comprising the protein of interest asa translational fusion with another protein or peptide which may beremovable by enzymatic or chemical cleavage. It is often observed in theproduction of certain peptides in recombinant systems that expression asa fusion protein prolongs the lifespan, increases the yield of thedesired peptide, or provides a convenient means of purifying the proteinof interest. A variety of peptidases (e.g. trypsin) which cleave apolypeptide at specific sites or digest the peptides from the amino orcarboxy termini (e.g. diaminopeptidase) of the peptide chain are known.Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave apolypeptide chain at specific sites. The skilled artisan will appreciatethe modifications necessary to the amino add sequence (and synthetic orsemi-synthetic coding sequence if recombinant means are employed) toincorporate site-specific internal cleavage sites. See e.g., P. Carter,“Site Specific Proteolysis of Fusion Proteins”, Chapter 13 in PROTEINPURIFICATION: FROM MOLECULAR MECHANISMS TO LARGE SCALE PROCESSES,American Chemical Society, Washington, D.C. (1990).

In addition to cloning and expressing the genes of interest in theprokaryotic systems discussed above, the proteins of the presentinvention may also be produced in eukaryotic systems. The presentinvention is not limited to use in a particular eukaryotic host cell. Avariety of eukaryotic host cells are available from depositories such asthe American Type Culture Collection (ATCC) and are suitable for usewith the vectors of the present invention. The choice of a particularhost cell depends to some extent on the particular expression vectorused to drive expression of the rhesus monkey neuropeptide Y-likereceptor-encoding nucleic acids of the present invention. Exemplary hostcells suitable for use in the present invention are listed in Table I

TABLE I Host Cell Origin Source HepG-2 Human Liver Hepatoblastoma ATCCHB 8065 CV-1 African Green Monkey Kidney ATCC CCL 70 LLC-MK₂ RhesusMonkey Kidney ATCC CCL 7 3T3 Mouse Embryo Fibroblasts ATCC CCL 92 CHO-K₁Chinese Hamster Ovary ATCC CCL 61 HeLa Human Cervix Epitheloid ATCC CCL2 RPMI8226 Human Myeloma ATCC CCL 155 H4IIEC3 Rat Hepatoma ATCC CCL 1600C127I Mouse Fibroblast ATCC CCL 1616 293 Human Embyronal Kidney ATCC CRL1573 HS-Sultan Human Plasma Cell Plasmocytoma ATCC CCL 1484 BHK-21 BabyHamster Kidney ATCC CCL 10

An especially preferred cell line employed in this invention is thewidely available cell line AV12-664 (hereinafter “AV12”). This cell lineis available from the American Type Culture Collection under theaccession number ATCC CRL 9595. The AV12 cell line was constructed byinjecting a Syrian hamster in the scruff of the neck with humanadenovirus 12 and isolating cells from the resulting tumor.

A wide variety of vectors, some of which are discussed below, exists forthe transformation of such mammalian host cells, but the specificvectors described herein are in no way intended to limit the scope ofthe present invention.

The pSV2-type vectors comprise segments of the simian virus 40 (SV40)genome that constitute a defined eukaryotic transcription unit-promoter,intervening sequence, and polyadenylation site. In the absence of theSV40 T antigen, the plasmid pSV2-type vectors transform mammalian andother eukaryotic host cells by integrating into the host cellchromosomal DNA. A large number of plasmid pSV2-type vectors have beenconstructed, such as plasmid pSV2-gpt, pSV2-neo, pSV2-dhfr, pSV2-hyg,and pSV2-β-globin, in which the SV40 promoter drives transcription of aninserted gene. These vectors are suitable for use with the codingsequences of the present invention and are widely available from sourcessuch as the ATCC or the Northern Regional Research Laboratory (NRRL),1815 N. University Street, Peoria, Ill., 61604.

The plasmid pSV2-dhfr (ATCC 37146) comprises a murine dihydrofolatereductase (dhfr) gene under the control of the SV40 early promoter.Under the appropriate conditions, the dhfr gene is known to beamplified, or copied, in the host chromosome. This amplification canresult in the amplification of closely-associated DNA sequences and can,therefore, be used to increase production of a protein of interest. See.e.g., J. Schinike, Cell, 35:705-713 (1984).

Plasmids constructed for expression of the proteins of the presentinvention in mammalian and other eukaryotic host cells can utilize awide variety of promoters. The present invention is in no way limited tothe use of the particular promoters exemplified herein. Promoters suchas the SV40 late promoter, promoters from eukaryotic genes, such as, forexample, the estrogen-inducible chicken ovalbumin gene, the interferongenes, the gluco-corticoid-inducible tyrosine aminotransferase gene, andthe thymidine kinase gene, and the major early and late adenovirus genescan be readily isolated and modified to express the genes of the presentinvention. Eukaryotic promoters can also be used in tandem to driveexpression of a coding sequence of this invention. Furthermore, a largenumber of retroviruses are known that infect a wide range of eukaryotichost cells. The long terminal repeats in the retroviral DNA frequentlyencode functional promoters and, therefore, may be used to driveexpression of the nucleic acids of the present invention.

Plasmid pRSVcat (ATCC 37152) comprises portions of a long terminalrepeat of the Rous Sarcoma virus, a virus known to infect chickens andother host cells. This long terminal repeat contains a promoter which issuitable for use in the vectors of this invention. H. Gorman, et al.,Proceedings of the National Academy of Sciences (USA), 79:6777 (1982).The plasmid pMSVi (NRRL B-15929) comprises the long terminal repeats ofthe Murine Sarcoma virus, a virus known to infect mouse and other hostcells.

The mouse metallothionein promoter has also been well characterized foruse in eukaryotic host cells and is suitable for use in the expressionof the nucleic acids of the present invention. The mouse metallothioneinpromoter is present in the plasmid pdBPV-MMTneo (ATCC 37224) which canserve as the starting material of other plasmids of the presentinvention.

An especially preferred expression vector system employs one of a seriesof vectors containing the BK enhancer, an enhancer derived from the BKvirus, a human papovavirus. The most preferred such vector systems arethose which employ not only the BK enhancer but also theadenovirus-2-early region 1A (E1A) gene product. The E1A gene product(actually, the E1A gene produces two products, which are collectivelyreferred to herein as “the E1A gene product”) is an immediate-early geneproduct of adenovirus, a large DNA virus.

A most preferred expression vector employed in the present invention isthe phd series of vectors which comprise a BK enhancer in tandem withthe adenovirus late promoter to drive expression of useful products ineukaryotic host cells. The construction and method of using the phdplasmid, as well as related plasmids, are described in U.S. Pat. No.5,242,688, issued Sep. 7, 1993, and 4,992,373, issued Feb. 12, 1991, allof which are herein incorporated by reference. Escerichia coli K12 GM48cells harboring the plasmid phd are available as part of the permanentstock collection of the Northern Regional Research Laboratory underaccession number NRRL B-18525. The plasmid may be isolated from thisculture using standard techniques.

The plasmid phd contains a unique BclI site which may be utilized forthe insertion of the gene encoding the protein of interest. The skilledartisan understands that linkers or adapters may be employed in cloningthe gene of interest into this BclI site. The phd series of plasmidsfunctions most efficiently when introduced into a host cell whichproduces the E1A gene product, cell lines such as AV12-664, 293 cells,and others, described supra.

Transformation of the mammalian cells can be performed by any of theknown processes including, but not limited to, the protoplast fusionmethod, the calcium phosphate co-precipitation method, electroporationand the like. See. e.g., J. Sambrook, et al., supra, at 3:16.30-3:16.66.

Other routes of production are well known to skilled artisans. Inaddition to the plasmid discussed above, it is well known in the artthat some viruses are also appropriate vectors. For example, theadenovirus, the adeno-associated virus, the vaccinia virus, the herpesvirus, the baculovirus, and the rous sarcoma virus are useful. Such amethod is described in U.S. Pat. No. 4,775,624, herein incorporated byreference. Several alternate methods of expression are described in J.Sambrook, et al., supra, at 16.3-17.44.

In addition to prokaryotes and mammalian host cells, eukaryotic microbessuch as yeast cultures may also be used. The imperfect fungusSaccharomyces cerevisiae, or common baker's yeast, is the most commonlyused eukaryotic microorganism, although a number of other strains arecommonly available. For expression in Saccharomyces sp., the plasmidYRp7 (ATCC-40053), for example, is commonly used. See. e.g., L.Stinchcomb,.et al., Nature (London), 282:39 (1979); J. Kingsman ., Gene,7:141 (1979); S. Tschemper . G, 10:157 (1980). This plasmid alreadycontains the trp gene which provides a selectable marker for a mutantstrain of yeast lacking the ability to grow in tryptophan.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase [found on plasmid pAP12BD (ATCC53231) and described in U.S. Pat. No. 4,935,350, issued Jun. 19, 1990,herein incorporated by reference] or other glycolytic enzymes such asenolase [found on plasmid pAC1 (ATCC 39532)], glyceraldehyde-3-phosphatedehydrogenase [derived from plasmid pHcGAPC1 (ATCC 57090, 57091)],hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase, as well as the alcohol dehydrogenase and pyruvatedecarboxylase genes of Zymomonas mobilis (U.S. Pat. No. 5,000,000 issuedMar. 19, 1991, herein incorporated by reference).

Other yeast promoters, which are inducible promoters, having theadditional advantage of their transcription being controllable byvarying growth conditions, are the promoter regions for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, metallothionein [contained onplasmid vector pCL28XhoLHBPV (ATCC 39475) and described in U.S. Pat. No.4,840,896, herein incorporated by reference], glyceraldehyde 3-phosphatedehydrogenase, and enzymes responsible for maltose and galactose [e.g.GAL1 found on plasmid pRY121 (ATCC 37658)] utilization. Suitable vectorsand promoters for use in yeast expression are further described in R.

Hitzeman et al., European Patent Publication No. 73,657A. Yeastenhancers such as the UAS Gal from Saccharomyces cervisiae (found inconjunction with the CYC1 promoter on plasmid YEpsec—hI1beta ATCC67024), also are advantageously used with yeast promoters.

Practitioners of this invention realize that, in addition to theabove-mentioned expression systems, the cloned cDNA may also be employedin the production of transgenic animals in which a test mammal, usuallya mouse, in which expression or overexpression of the proteins of thepresent invention can be assessed. The nucleic acids of the presentinvention may also be employed in the construction of “knockout” animalsin which the expression of the native cognate of the gene is suppressed.

Skilled artisans also recognize that some alterations of SEQ ID NO:2will fail to change the function of the amino acid compound. Forinstance, some hydrophobic amino acids may be exchanged for otherhydrophobic amino acids. Those altered amino acid compounds which confersubstantially the same function in substantially the same manner as theexemplified amino acid compound are also encompassed within the presentinvention. Typical such conservative substitutions attempt to preservethe: (a) secondary or tertiary structure of the polypeptide backbone;(b) the charge or hydrophobicity of the residue; or (c) the bulk of theside chain. Some examples of such conservative substitutions of aminoacids, resulting in the production of proteins which are functionalequivalents of the protein of SEQ ID NO:2 are shown in Table II, infra.

TABLE II Original Residue Exemplary Substitutions Ala Ser, Gly Arg LysAsn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro, Ala His Asn, GlnIle Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Mel Leu, Ile Phe Met, Leu,Gyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

These substitutions may be introduced into the protein in a variety ofways, such as during the chemical synthesis or by chemical modificationof an amino acid side chain after the protein has been prepared.

Alterations of the protein having a sequence which corresponds to thesequence of SEQ ID NO:2 may also be induced by alterations of thenucleic acid compounds which encodes these proteins. These mutations ofthe nucleic acid compound may be generated by either random mutagenesistechniques, such as those techniques employing chemical mutagens, or bysite-specific mutagenesis employing oligonucleotides. Those nucleic acidcompounds which confer substantially the same function in substantiallythe same manner as the exemplified nucleic acid compounds are alsoencompassed within the present invention.

Other embodiments of the present invention are nucleic acid compoundswhich comprise isolated nucleic acid sequences which encode SEQ ID NO:2.As skilled artisans will recognize, the amino acid compounds of theinvention can be encoded by a multitude of different nucleic acidsequences because most of the amino acids are encoded by more than onenucleic acid triplet due to the degeneracy of the amino acid code.Because these alternative nucleic acid sequences would encode the sameamino acid sequences, the present invention further comprises thesealternate nucleic acid sequences.

The gene encoding the rhesus monkey Y2 receptor molecule may be producedusing synthetic methodology. This synthesis of nucleic acids is wellknown in the art. See, e.g., E. L. Brown, R. Belagaje, M. J. Ryan, andH. G. Khorana, Methods in Enzymology, 68:109-151 (1979). The DNAsegments corresponding to the receptor gene are generated usingconventional DNA synthesizing apparatus such as the Applied BiosystemsModel 380A or 380B DNA synthesizers (commercially available from AppliedBiosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) whichemploy phosphoramidite chemistry. In the alternative, the moretraditional phosphotriester chemistry may be employed to synthesize thenucleic acids of this invention. See e.g., M. J. Gait, ed.,OLIGONUCLEOTIDE SYNTHESIS, A PRACTICAL APPROACH, (1984).

The synthetic rhesus monkey Y2 receptor gene may be designed to possessrestriction endonuclease cleavage sites at either end of the transcriptto facilitate isolation from and integration into expression andamplification plasmids. The choice of restriction sites are chosen so asto properly orient the coding sequence of the receptor with controlsequences to achieve proper in-frame reading and expression of the Y2receptor molecule. A variety of other such cleavage sites may beincorporated depending on the particular plasmid constructs employed andmay be generated by techniques well known in the art.

In an alternative methodology, the desired DNA sequences can begenerated using the polymerase chain reaction as described in U.S. Pat.No. 4,889,818, which is herein incorporated by reference.

In addition to the deoxyribonucleic acid of SEQ ID NO:1, this inventionalso provides ribonucleic acids (RNA) which comprise the RNA sequence

1 UGAUUGAGGU ACAAGUUGUA GACUCUUGUG CUGGUUGCAG GCCAAGUGGA 51 ACUGUACUGAAAAUGGGUCC AAUAGGUACA GAGGCUGAUG AGAACCAGAC 101 AGUGGAAGAA AUGAAGGUGGAACAAUAUGG GCCACAAACC ACUCCUAGAG 151 GUGAACUGGU CCCUGAUCCU GAGCCAGAGCUUAUAGAUAG UACCAAGCUG 201 AUUGAGGUAC AAGUUGUCCU CAUAUUGGCC UAUUGCUCCAUCAUCUUGCU 251 UGGGGUAAUU GGCAACUCCU UGGUGAUCCA CGUGGUGAUC AAAUUCAAGA301 GCAUGCGCAC AGUAACCAAC UUUUUCAUCG CCAAUCUGGC UGUGGCAGAU 351CUUGUGGUGA AUACUCUGUG UCUACCAUUC ACUCUUACCU ACACCUUAAU 401 GGGGGAGUGGAAAAUGGGUC CUGUCCUGUG CCACCUGGUG CCCUAUGCAC 451 AGGGCCUGGC AGUACAAGUAUCCACAAUCA CCUUGACAGU AAUUGCCCUG 501 GACCGGCACA GGUGCAUCGU CUACCACCUGGAGAGCAAGA UCUCCAAGCG 551 UAUCAGCUUC CUGAUUAUUG GCUUGGCCUG GGGCAUCAGUGCCCUGCUAG 601 CAAGUCCCCU GGCCAUCUUC CGGGAGUAUU CACUGAUUGA GAUCAUUCCG651 GAUUUUGAGA UUGUGGCCUG UACUGAAAAA UGGCCUGGCG AGGAAAAGAG 701CAUCUAUGGC ACUGUCUACA GUCUUUCUUC CUUGUUGAUC CUGUACGUUU 751 UGCCUCUGGGCAUAAUAUCA UUUUCCUACA CUCGCAUUUG GAGUAAAUUG 801 AAGAGCCAUG UCAGUCCUGGAGCUGCAAAU GACCACUACC AUCAGCGAAG 851 GCAAAAAACC ACCAAAAUGC UGGUGUGCGUGGUGGUGGUG UUUGCGGUCA 901 GCUGGCUGCC UCUCCAUGCC UUCCAGCUUG CCGUUGACAUUGACAGCCAU 951 GUCCUGGACC UGAAGGAGUA CAAACUCAUC UUCACAGUGU UCCACAUCAU1001 CGCCAUGUGC UCCACUUUUG CCAAUCCCCU UCUCUAUGGC UGGAUGAACA 1051GCAACUAUAG AAAGGCUUUC CUCUCUGCCU UCCGCUGUGA GCAGCGGUUG 1101 GAUGCCAUUCACUCUGAGGU GUCCGUGACA UUCAAGGCUA AAAAGAACCU 1151 GGAGGUCAGA AAAAAUAGUGGCCCCAAUGA CUCUUUCACA GAAGCUACCA 1201 AUGUCUAAGG AAGCUAGGGU GUGAAAAUGUAUGAAUGAAU UCUGACCAGA 1251 GCUAUAAAUC UGGUUGAUGG CGGCUCACAA GUGAUAAUUGAUUUCCCAUU 1301 UUAAGGAAGA AGAGGAUCUA AAUGGAAGCA UCUGCUGUUU AGUUCCUGGA1351 AAACUGGCUG GGAAGAGCCU GUGUGAAAAU ACUUGAAUUC AAAGAUAAGG 1401CAGCAAAAUG GUUUACUUAA CAGUUGGUAG GGUAGUAGGU UGAAUUAGGA 1451 GUAAAAGCAGAGAGAGGUAC UUUUGACUAU UUUCCUGGAG UGAAGUAAAC 1501 UUGAACAAGG AAUUGGUAUUAUCAGCAUUG CAAAGAGACG GUGGGUAAAU 1551 AAGUUGAUUU UCAGAUUUCA UUAGGACCUGGAUUGGGGAG CUGUGUAGUU 1601 CACGGUUCCC UGCUUGGCUG AUGAAAACGU CGCUGAACAAAAAUUUCUCC 1651 AGGGAGCCAC AGGCUCUCCU UCAUCACGUU UUGAUUUUUU UUGUUAAUUC1701 UCUAGACAAA AUCCAUCAAG GAAUGCUGCA CGAAAAGAUU GCCAGCUAUA 1751UGAAUGGCUU CAAGGAACUA AACUGAAACU UGCUAUAUAA UUAAUAUUUU 1801 GGCACACGAUAGGGGAACUC CUCAACACUC AGUGAGCCAA UUGUUCUUAA 1851 AACCGGUUGC ACAUUUGGUGAAAGUUUCUU CAACUCUGAA UCAAAACCUG 1901 AAAUUCUCAC AAUUGCAGGA AAUGCAAACCAUCAUUUAAU UUGUAAUUUC 1951 AAGUUACAUC UGCUUUAUGG AGAUAUUUAG AUAACAAGCAUACAACUUGA 2001 UAGUUUUAUU GUUAUACCUU UUUGAACAUG UAUGAUUUAU GUUAUUAUUC2051 CUAUUGGACC UAAGUUUGUC UACACUAAAA UUUAAAUCAC AAUAAAGAAU 2101AAUUUUUGUG GAAAAAAAAA AAAAAAAAAA AAAAAAAACU CGAG

hereinafter referred to as SEQ ID NO:3, or the complementary ribonucleicacid, or a fragment of either SEQ ID NO:3 or the complement thereof. Theribonucleic acids of the present invention may be prepared using thepolynucleotide synthetic methods discussed supra or they may be preparedenzymatically using RNA polymerases to transcribe a DNA template.complement thereof.

The most preferred systems for preparing the ribonucleic acids of thepresent invention employ the RNA polymerase from the bacteriophage T7 orthe bacteriophage SP6. Both of these RNA polymerases are highly specificand require the insertion of bacteriophage-specific sequences at the5′end of the message to be read. S, J. Sambrook, et al., supra, at18.82-18.84.

This invention also provides nucleic acids, RNA or DNA, which arecomplementary to SEQ ID NO:1 or SEQ ID NO:3.

The present invention also provides probes and primers useful formolecular biology techniques. A compound which encodes for SEQ ID NO:1,SEQ ID NO:3 or a complementary sequence of SEQ ID NO:1 or SEQ ID NO:3,or a fragment thereof, and which is at least 18 base pairs in length,and which will selectively hybridize to rhesus monkey genomic DNA ormessenger RNA encoding a rhesus monkey neuropeptide Y receptor, isprovided. Preferably, the 18 or more base pair compound is DNA.

The term “selectively hybridize” as used herein may refer to either oftwo situations. In the first such embodiment of this invention, thenucleic acid compounds described supra hybridize to a rhesus monkeyneuropeptide Y receptor under more stringent hybridization conditionsthan these same nucleic acid compounds would hybridize to an analogousneuropeptide Y receptor of another species, e.g. murine or primate. Inthe second such embodiment of this invention, these probes hybridize tothe Y2 receptor under more stringent hybridization conditions than otherrelated compounds, including nucleic acid sequences encoding otherneuropeptide Y receptors.

These probes and primers can be prepared enzymatically as describedsupra. In a most preferred embodiment these probes and primers aresynthesized using chemical means as described supra. Probes and primersof defined structure may also be purchased commercially.

This invention also encompasses recombinant DNA cloning vectors andexpression vectors comprising the nucleic acids of the presentinvention. Many of the vectors encompassed within this invention aredescribed above. The preferred nucleic acid vectors are those which areDNA. The most preferred recombinant DNA vector comprises the isolatedDNA sequence SEQ ID NO:1.

Any plasmid comprising the gene of the present invention is readilymodified to construct expression vectors that produce Y2 receptors in avariety of organisms, including, for example, E. coli, Sf9 (as host forbaculovirus), Spodoptera and Saccharomyces. The current literaturecontains techniques for constructing AV12 expression vectors and fortransforming AV12 host cells. U.S. Pat. No. 4,992,373, hereinincorporated by reference, is one of many references describing thesetechniques.

One of the most widely employed techniques for altering a nucleic acidsequence is by way of oligonucleotide-directed site-specificmutagenesis. B. Comack, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,8.01-8.5.9, (F. Ausubel, et al, eds. 1991). In this technique anoligonucleotide, whose sequence contains the mutation of interest, issynthesized as described supra. This oligonucleotide is then hybridizedto a template containing the wild-type sequence. In a most preferredembodiment of this technique, the template is a single-strandedtemplate. Particularly preferred are plasmids which contain regions suchas the f1 intergenic region. This region allows the generation ofsingle-stranded templates when a helper phage is added to the cultureharboring the “phagemid”.

After the annealing of the oligonucleotide to the template, aDNA-dependent DNA polymerase is then used to synthesize the secondstrand from the oliognucleotide, complementary to the template DNA. Theresulting product is a heteroduplex molecule containing a mismatch dueto the mutation in the oligonucleotide. After DNA replication by thehost cell a mixture of two types of plasmid are present, the wild-typeand the newly constructed mutant. This technique permits theintroduction of convenient restriction sites such that the codingsequence may be placed immediately adjacent to whichever transcriptionalor translational regulatory elements are employed by the practitioner.

The construction protocols utilized for E. coli can be followed toconstruct analogous vectors for other organisms, merely by substituting,if necessary, the appropriate regulatory elements using techniques wellknown to skilled artisans.

Host cells which harbor the nucleic acids provided by the presentinvention are also provided. A preferred host cell is an Xenopus sp.oocyte which has been injected with RNA or DNA compounds of the presentinvention. Most preferred oocytes of the present invention are thosewhich harbor a sense mRNA of the present invention. Other preferred hostcells include AV12 and E. coli cells which have been transfected and/ortransformed with a vector which comprises a nucleic acid of the presentinvention.

The present invention also provides a method for constructing arecombinant host cell capable of expressing SEQ ID NO:2, said methodcomprising transforming a host cell with a recombinant DNA vector thatcomprises an isolated DNA sequence which encodes SEQ ID NO:2. Thepreferred host cell is AV12. The preferred vector for expression is onewhich comprises SEQ ID NO:1. Another preferred host cell for this methodis E. coli. An especially preferred expression vector in E. coli is onewhich comprises SEQ ID NO:1. Transformed host cells may be culturedunder conditions well known to skilled artisans such that SEQ ID NO:2 isexpressed, thereby producing the Y2 receptor in the recombinant hostcell.

The ability of neuropeptide Y, pancreatic polypeptide, and peptide YY,to bind to the Y2 receptor is essential in the development of amultitude of indications. In developing agents which act as antagonistsor agonists of the Y2 receptor, it would be desirable, therefore, todetermine those agents which bind the Y2 receptor. Generally, such anassay includes a method for determining whether a substance is afunctional ligand of the Y2 receptor, said method comprising contactinga functional compound of the Y2 receptor with said substance, monitoringbinding activity by physically detectable means, and identifying thosesubstances which effect a chosen response. Preferably, the physicallydetectable means is competition with labeled neuropeptide Y (orpancreatic polypeptide, or peptide YY) or binding of ligand in an oocytetransient expression system

The instant invention provides such a screening system useful fordiscovering agents which compete with neuropeptide Y for binding to theY2 receptor, said screening system comprising the steps of:

a) isolating a rhesus monkey Y2 receptor;

b) exposing said rhesus monkey Y2 receptor to a potential inhibitor orsurrogate of the neuropeptide Y/Y2 receptor complex;

c) introducing neuropeptide Y (or pancreatic polypeptide or peptide YY);

d) removing non-specifically bound molecules; and

e) quantifying the concentration of bound potential inhibitor and/orneuropeptide Y (or pancreatic polypeptide or peptide YY).

This allows one to rapidly screen for inhibitors or surrogates of theformation of the neuropeptide Y/Y2 receptor complex. Utilization of thescreening system described above provides a sensitive and rapid means todetermine compounds which interfere with the formation of theneuropeptide Y/Y2 receptor complex. This screening system may also beadapted to automated procedures such as a PANDEX® (Baxter-DadeDiagnostics) system allowing for efficient high-volume screening ofpotential therapeutic agents.

In the assay supra, as well those infra, the neuropeptide Y employedtherein may be replaced with pancreatic polypeptide or peptide YY. Theneuropeptide Y used as a ligand is, therefore, merely illustrative andis not to be considered limiting in any way.

In such a screening protocol a Y2 receptor is prepared as elsewheredescribed herein, preferably using recombinant DNA technology. A sampleof a test compound is then introduced to the reaction vessel containingthe Y2 receptor followed by the addition of neuropeptide Y (orpancreatic polypeptide or peptide YY). In the alternative theneuropeptide Y (or pancreatic polypeptide or peptide YY) may be addedsimultaneously with the test compound. Unbound molecules are washed freeand the eluent inspected for the presence of neuropeptide Y (orpancreatic polypeptide or peptide YY) or the test compound.

For example, in a preferred method of the invention, radioactively orchemically labeled neuropeptide Y (or pancreatic polypeptide or peptideYY) may be used. The eluent is then scored for the chemical label orradioactivity. The absence or diminution of the chemical label orradioactivity indicates the formation of the neuropeptide Y/Y2 receptorcomplex. This indicates that the test compound has not effectivelycompeted with neuropeptide Y in the formation of the neuropeptide Y/Y2receptor complex. The presence of the chemical label or radioactivityindicates that the test compound has competed with neuropeptide Y in theformation of the neuropeptide Y/Y2 receptor complex. Similarly, aradioactively or chemically labeled test compound may be used in whichcase the same steps as outlined above would be used except that theinterpretation of results would be the converse of using radioactivelyor chemically labeled neuropeptide Y.

As would be understood by the skilled artisan these assays may also beperformed such that the practitioner measures the radioactivity orfluorescence remaining with the protein, not in the eluent. A preferredsuch assay employs radiolabeled neuropeptide Y (or pancreaticpolypeptide or peptide YY). After the competition reaction has beenperformed the reaction mixture is then passed through a filter, thefilter retaining the receptor and whatever is complexed with thereceptor. The radioactivity on each filter is then measured in ascintillation counter. In such an assay higher amounts of radiolabelpresent indicate lower affinity for the receptor by the test compound.

The Y2 receptor may be free in solution or bound to a solid support.Whether the Y2 receptor is bound to a support or is free in solution, itis generally important that the conformation of the protein beconserved. In a preferred practice of the invention, therefore, the Y2receptor is suspended in a hydrophobic environment employing natural orsynthetic detergents, membrane suspensions, and the like. Preferreddetergent complexes include the zwitterionic detergent3-[(3-cholamidopropyl)-dimethylammonio ]-1-propane sulfonate (“CHAPS”)as well as sodium deoxycholate.

Skilled artisans will recognize that desirable dissociation constant(K_(i)) values are dependent on the selectivity of the compound tested.For example, a compound with a K_(i) which is less than 10 nM isgenerally considered an excellent candidate for drug therapy. However, acompound which has a lower affinity, but is selective for the particularreceptor, may be an even better candidate. The present invention,however, provides radiolabeled competition assays, whether resultstherefrom indicate high affinity or low affinity to Y2 receptor, becauseskilled artisans will recognize that any information regarding bindingor selectivity of a particular compound is beneficial in thepharmaceutical development of drugs.

Assays useful for evaluating neuropeptide Y receptor antagonists arewell known in the art. See, e.g., U.S. Pat. No. 5,284,839, issued Feb.8, 1994, which is herein incorporated by reference. See also, M. W.Walker, et al., Journal of Neurosciences, 8:2438-2446 (1988).

Transient Transfection Protocol

Cos-1 cells are seeded at a density of 10⁵ cells/150 mm dish on day one.On day three the cells are transfected (using commercially availablekits) with either 25 μg rhesus monkey Y2 receptor, 50 iug rhesus monkeyY2 receptor, or 25 iug pSVLuc (SV40 Luciferase-control). Briefly, 4 mlmedia containing supercoiled DNA are combined with 4 ml media containing0.6 ml of the commercial tranfection enhancing agent while mixing. Thismixture is incubated for 15 minutes at room temperature, then 16 ml ofmedia is added to the tube and gently mixed. The cells are washed withPBS and 10 ml is added per dish. The cells are incubated at 37° C. for 6hours and 10 ml of media containing 20% fetal bovine serum is added. Onday 5 (48 hrs post-transfection) the cells are scraped into phosphatebuffered saline, pelleted, and kept on ice until binding assays areperformed.

Stable Transfection of CHO Cells

A vector containing the Y2 receptor insert, is linearized using andtransfected into Chinese hamster ovary (CHO) cells using commerciallyavailable reagents. The cells are maintained under 5% carbon dioxide inDulbecco's Modified Eagle's Medium (DMEM)/Ham's F-12 Medium (3:1)containing 10% fetal bovine serum, 2 mM glutamine, 100 internationalunits of penicillin, and 100 μg/ml streptomycin. Stably transfectedcells are selected with 500 μg/ml G418 and tested for their ability tobind [¹²⁵I]-PYY, infra.

[¹²⁵I]-PYY Binding Protocol

The homogenate binding studies are conducted using known methods. See.e.g. D.R. Gehlert, et al., Neurochemistry International, 21: 45-67(1992). The cell pellets are resuspended using a glass homogenizer in 25mM HEPES (pH 7.4) buffer containing 2.5 mM calcium chloride, 1 mMmagnesium chloride and 2 g/l Bacitracin. Incubations are performed in afinal volume of 200 μl containing various concentrations [¹²⁵I]-PYY (SA2200 Ci/mmol) or [¹²⁵I]-PYY (SA 2000 Ci/mmol) and 0.2-0.4 mg protein for2 hours at room temperature. Nonspecific binding is defined as theamount of radioactivity remaining bound to the tissue after incubatingin the presence of 1 μM hPP.

In pharmacological studies, various concentrations of peptides areincluded in the incubation mixture. Saturation experiments are performedwith each radioligand by incubating in various concentrations of theradioligand in the assay. Incubations are terminated by rapid filtrationthrough glass fiber filters, which had been presoaked in 0.3%polyethyleneimine, using a cell harvester. The filters are washed with 5ml of 50 mM Tris (pH 7.4) at 4° C. and rapidly dried at 60° C. The driedfilters are treated with melt-on scintillator sheets, and theradioactivity retained on the filters are counted. The results areanalyzed using the Lundon-1 software package (Lundon Inc., ChagrinFalls, Ohio) running on a VAX computer or the Cheng-Prushoff equation.Protein concentrations are measured using standard staining techniques,using bovine serum albumin for standards.

In one such competition assay, a battery of known neuropeptide Yreceptor antagonists, agonists, and partial agonists are evaluated fortheir relative abilities to inhibit the binding of [¹²⁵I]peptide YY tothe rhesus monkey Y2 receptor of the present invention.

The previously described screening systems identify compounds whichcompetitively bind to the Y2 receptor. Determination of the ability ofsuch compounds to stimulate or inhibit the action of the Y2 receptor isessential to further development of such compounds for therapeuticapplications. The need for a bioactivity assay system which determinesthe response of the Y2 receptor to a compound is clear. The instantinvention provides such a bioactivity assay, said assay comprising thesteps of:

a) transfecting a mammalian host cell with an expression vectorcomprising DNA encoding a Y2 receptor;

b) culturing said host cell under conditions such that the DNA encodingthe Y2 receptor is expressed,

c) exposing said host cell so transfected to a test compound, and

d) measuring the change in a physiological condition known to beinfluenced by the binding of neuropeptide Y to the Y2 receptor relativeto a control in which the transfected host cell is exposed toneuropeptide Y.

An oocyte transient expression system can be constructed according tothe procedure described in S. Lübbert, et al., Proceedings of theNational Academy of Sciences (USA), 84:4332 (1987).

In an especially preferred embodiment of this invention an assaymeasuring the inhibition of forskolin-stimulated cAMP synthesis isperformed. The inhibition of cAMP synthesis is known to positivelycorrelated with the addition of neuropeptide Y to cells containingcertain types of neuropeptide Y receptors.

Adenylate Cyclase Activity.

Adenylate cyclase activity is determined in initial experiments intransfected mammalian cells, using standard techniques. See, e.g., N.Adham, et al., supra,; R. L. Weinshank, et al., Proceedings of theNational Academy of Sciences (USA), 89:3630-3634 (1992), and thereferences cited therein.

Adenylate cyclase activity is measured using CHO cells stablytransfected with the Y2 receptor and prelabelled with 2μCi/ml of[³H]-adenine for three hours. The media is removed and replaced withincubation media containing a Tyrode-HEPES buffer, 100 μM Rolipram, 10μM indolidan, 10 μM phosphoramidon and 12.6 mg bacitracin/100 ml.Adenylate cyclase activity is increased by the addition of 15 μMforskolin and the ability of the peptide to inhibit this activation ismeasured using various concentrations. Following the incubation, the[³H]-cAMP is separated by chromatography on alumina columns with[³²p]-cAMP as an internal standard. The results are quantified by dualchannel scintillation counting.

In another embodiment of this invention an assay which correlatesneuropeptide Y activity with the hydrolysis of phosphatidylinositol isperformed. The hydrolysis of phosphatidylinositol is known to positivelycorrelate with addition of neuropeptide Y. This biochemical assay isperformed essentially as described by M. Berridge, Biochemistry Journal,212:849 (1983).

Phosphatidylinositol Assay

Twenty-four-well tissue-culture vessels are seeded with approximately250,000 cells per well in Dulbecco's Minimal Essential Media (D-MEM) (inthe absence of glutamic acid) which contained 2 mM glutamine and 10%dialyzed fetal calf serum. After 24 hours growth at 37° C. the media wasremoved and replaced with fresh media containing four microcuries of[³H]myoinositol per well and the cultures are incubated a further 16 to20 hours. The media was then removed and the cells in each well arewashed with serum free medium containing 10 mM lithium chloride, 10 mMmyoinositol, and 10 mM HEPES (2×1 ml washes). After the final wash, 0.5ml of washing solution was added containing the appropriateconcentrations of drugs and vehicles.

If the particular assay was testing antagonists, a ten minute 10incubation was performed prior to agonist induction. Cells are incubatedfor about one hour at 37° C. in 95%/5% O₂/CO₂ or as appropriate for timecourse. The reactions are terminated by removing media and adding 1 mlof cold 1:1 acetone:methanol followed by induction on ice for a minimumof twenty minutes.

These extracts are then removed and placed in 1.5 ml centrifuge tubes.Each well was washed with 0.5 ml water and this wash was added to theappropriate extract. After mixing and centrifugation, each aqueoussupernatant was processed by chromatography on a QMA SEP-PAK® column,which had previously been wetted and equilibrated by passing 10 ml ofwater, followed by 8 ml of 1 M triethylammonium hydrogen carbonate(TEAB), followed by 10 ml of water through the column.

The assay supernatants containing the water soluble [³H]inositolphosphate are passed over the columns. This was followed by a 10 mlwater wash and a 4 ml wash with 0.02 MTEAB to remove [³H]inositolprecursors [³H]Inositol phosphate was eluted with 4 ml of 0.1 M TEABinto scintillation vials and counted in the presence of scintillationcocktail. Total protein in each sample was measured using standardtechniques. Assays are measured as the amount of [³H]inositol phosphaterelease per milligram of protein.

In another embodiment this invention provides a method for identifying,in a test sample, DNA homologous to a probe of the present invention,wherein the test nucleic acid is contacted with the probe underhybridizing conditions and identified as being homologous to the probe.Hybridization techniques are well known in the art. See. e.g., J.Sambrook, et al., supra, at Chapter 11.

The nucleic acid compounds of the present invention may also be used tohybridize to genomic DNA which has been digested with one or morerestriction enzymes and run on an electrophoretic gel. The hybridizationof radiolabeled probes onto such restricted DNA, usually fixed to amembrane after electrophoresis, is well known in the art. See. e.g., J.Sambrook, supra. Such procedures may be employed in searching forpersons with mutations in these receptors by the well-known techniquesof restriction fragment length polymorphisms (RFLP), the procedures ofwhich are described in U.S. Pat. No. 4,666,828, issued May 19, 1987, theentire contents of which is herein incorporated by reference.

The proteins of this invention as well as fragments of these proteinsmay be used as antigens for the synthesis of antibodies. The term“antibody” as used herein describes antibodies, fragments of antibodies(such as, but not limited, to Fab, Fab′, Fab₂′, and Fv fragments), andchimeric, humanized, veneered, resurfaced, or CDR-grafted antibodiescapable of binding antigens of a similar nature as the parent antibodymolecule from which they are derived. The instant invention alsoencompasses single chain polypeptide binding molecules.

The term “antibody” as used herein is not limited by the manner in whichthe antibodies are produced, whether such production is in situ or not.The term “antibody” as used in this specification encompasses thoseantibodies produced by recombinant DNA technology means including, butnot limited, to expression in bacteria, yeast, insect cell lines, ormammalian cell lines.

The production of antibodies, both monoclonal and polydonal, in animals,especially mice, is well known in the art. See, e.g., C. Milstein,HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, (Blackwell Scientific Pub., 1986);J. Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, (AcademicPress, 1983). For the production of monoclonal antibodies the basicprocess begins with injecting a mouse, or other suitable animal, with animmunogen. The mouse is subsequently sacrificed and cells taken from itsspleen are fused with myeloma cells, resulting in a hybridoma thatreproduces in vitro. The population of hybridomas is screened to isolateindividual clones, each of which secretes a single antibody species,specific for the immunogen. The individual antibody species obtained inthis way is each the product of a single B cell from the immune animalgenerated in response to a specific antigenic site, or epitope,recognized on the immunogenic substance.

Chimeric antibodies are described in U.S. Pat. No. 4,816,567, whichissued Mar. 28, 1989 to S. Cabilly, e. This reference discloses methodsand vectors for the preparation of chimeric antibodies. The entirecontents of U.S. Pat. No. 4,816,567 are herein incorporated byreference. An alternative approach to production of geneticallyengineered antibodies is provided in U.S. Pat. No. 4,816,397, which alsoissued Mar. 28, 1989 to M. Boss, et al. , the entire contents of whichare herein incorporated by reference. The Boss patent teaches thesimultaneous co-expression of the heavy and light chains of the antibodyin the same host cell.

The approach of U.S. Pat.No. 4,816,397 has been further refined astaught in European Patent Publication No. 0 239 400, which publishedSep. 30, 1987. The teachings of this European patent publication(Winter) are a preferred format for the genetic engineering of thereactive monoclonal antibodies of this invention. The Winter technologyinvolves the replacement of complementarity determining regions (CDRs)of a human antibody with the CDRs of a murine monoclonal antibodythereby converting the specificity of the human antibody to thespecificity of the murine antibody which was the source of the CDRregions. This “CDR grafting” technology affords a molecule containingminimal murine sequence and thus is less immunogenic.

Single chain antibody technology is yet another variety of geneticallyengineered antibody which is now well known in the art. See, e.g. R. E.Bird, et al., Science242:423-426 (1988); PCT Publication No. WO88/01649, which was published Mar. 10, 1988; U.S. Pat. No. 5,260,203,issued Nov. 9, 1993, the entire contents of which are hereinincorporated by reference. The single chain antibody technology involvesjoining the binding regions of heavy and light chains with a polypeptidesequence to generate a single polypeptide having the binding specificityof the antibody from which it was derived.

The aforementioned genetic engineering approaches provide the skilledartisan with numerous means to generate molecules which retain thebinding characteristics of the parental antibody while affording a lessimmunogenic format.

These antibodies are used in diagnostics, therapeutics or indiagnostic/therapeutic combinations. By “diagnostics” as used herein ismeant testing that is related to either the in vitro or in vivodiagnosis of disease states or biological status in mammals, preferablyin humans. By “therapeutics” and “therapeutic/diagnostic combinations”as used herein is respectively meant the treatment or the diagnosis andtreatment of disease states or biological status by the in vivoadministration to mammals, preferably humans, of the antibodies of thepresent invention. The antibodies of the present invention areespecially preferred in the diagnosis and/or treatment of conditionsassociated with an excess or deficiency of Y2 receptors.

In addition to being functional as direct therapeutic and diagnosticaids, the availability of a family of antibodies which are specific forthe Y2 receptor enables the development of numerous assay systems fordetecting agents which bind to this receptor. One such assay systemcomprises radiolabeling Y2 receptor-specific antibodies with aradionuclide such as ¹²⁵I and measuring displacement of the radiolabeledY2 receptor-specific antibody from solid phase Y2 receptor in thepresence of a potential antagonist.

Numerous other assay systems are also readily adaptable to detect agentswhich bind Y2 receptor. Examples of these aforementioned assay systemsare discussed in Methods in Enzymology, (J. Langone. and H. Vunakis,eds. 1981), Vol. 73, Part B, the contents of which are hereinincorporated by reference. Skilled artisans are directed to Section IIof Methods in Enzymology, Vol. 73, Part B, supra, which discusseslabeling of antibodies and antigens, and Section IV, which discussesimmunoassay methods.

In addition to the aforementioned antibodies specific for the Y2receptor, this invention also provides antibodies which are specific forthe hypervariable regions of the anti-Y2 receptor antibodies. Some suchanti-idiotypic antibodies would resemble the original epitope, the Y2receptor, and, therefore, would be useful in evaluating theeffectiveness of compounds which are potential antagonists, agonists, orpartial agonists of the Y2 receptor. See. e.g., Cleveland, et al, Nature(London), 305:56 (1983); Wasserman, et al, Proceedings of the NationalAcademy of Sciences (USA), 79:4810 (1982).

In another embodiment, this invention encompasses pharmaceuticalformulations for parenteral administration which contain, as the activeingredient, the anti-Y2 receptor antibodies described, supra. Suchformulations are prepared by methods commonly used in pharmaceuticalchemistry.

Products for parenteral administration are often formulated anddistributed in solid, preferably freeze-dried form, for reconstitutionimmediately before use. Such formulations are useful compositions of thepresent invention. Their preparation is well understood bypharmaceutical chemists.

In general, these formulations comprise the active ingredient incombination with a mixture of inorganic salts, to confer isotonicity, aswell as dispersing agents such as lactose, to allow the driedpreparation to dissolve quickly upon reconstitution. Such formulationsare reconstituted for use with highly purified water to a knownconcentration.

Alternatively, a water soluble form of the antibody can be dissolved inone of the commonly used intravenous fluids and administered byinfusion. Such fluids include physiological saline, Ringer's solution ora 5% dextrose solution.

3 2144 base pairs nucleic acid single linear cDNA CDS 63..1205 1TGATTGAGGT ACAAGTTGTA GACTCTTGTG CTGGTTGCAG GCCAAGTGGA ACTGTACTGA 60 AAATG GGT CCA ATA GGT ACA GAG GCT GAT GAG AAC CAG ACA GTG GAA 107 Met GlyPro Ile Gly Thr Glu Ala Asp Glu Asn Gln Thr Val Glu 1 5 10 15 GAA ATGAAG GTG GAA CAA TAT GGG CCA CAA ACC ACT CCT AGA GGT GAA 155 Glu Met LysVal Glu Gln Tyr Gly Pro Gln Thr Thr Pro Arg Gly Glu 20 25 30 CTG GTC CCTGAT CCT GAG CCA GAG CTT ATA GAT AGT ACC AAG CTG ATT 203 Leu Val Pro AspPro Glu Pro Glu Leu Ile Asp Ser Thr Lys Leu Ile 35 40 45 GAG GTA CAA GTTGTC CTC ATA TTG GCC TAT TGC TCC ATC ATC TTG CTT 251 Glu Val Gln Val ValLeu Ile Leu Ala Tyr Cys Ser Ile Ile Leu Leu 50 55 60 GGG GTA ATT GGC AACTCC TTG GTG ATC CAC GTG GTG ATC AAA TTC AAG 299 Gly Val Ile Gly Asn SerLeu Val Ile His Val Val Ile Lys Phe Lys 65 70 75 AGC ATG CGC ACA GTA ACCAAC TTT TTC ATC GCC AAT CTG GCT GTG GCA 347 Ser Met Arg Thr Val Thr AsnPhe Phe Ile Ala Asn Leu Ala Val Ala 80 85 90 95 GAT CTT GTG GTG AAT ACTCTG TGT CTA CCA TTC ACT CTT ACC TAC ACC 395 Asp Leu Val Val Asn Thr LeuCys Leu Pro Phe Thr Leu Thr Tyr Thr 100 105 110 TTA ATG GGG GAG TGG AAAATG GGT CCT GTC CTG TGC CAC CTG GTG CCC 443 Leu Met Gly Glu Trp Lys MetGly Pro Val Leu Cys His Leu Val Pro 115 120 125 TAT GCA CAG GGC CTG GCAGTA CAA GTA TCC ACA ATC ACC TTG ACA GTA 491 Tyr Ala Gln Gly Leu Ala ValGln Val Ser Thr Ile Thr Leu Thr Val 130 135 140 ATT GCC CTG GAC CGG CACAGG TGC ATC GTC TAC CAC CTG GAG AGC AAG 539 Ile Ala Leu Asp Arg His ArgCys Ile Val Tyr His Leu Glu Ser Lys 145 150 155 ATC TCC AAG CGT ATC AGCTTC CTG ATT ATT GGC TTG GCC TGG GGC ATC 587 Ile Ser Lys Arg Ile Ser PheLeu Ile Ile Gly Leu Ala Trp Gly Ile 160 165 170 175 AGT GCC CTG CTA GCAAGT CCC CTG GCC ATC TTC CGG GAG TAT TCA CTG 635 Ser Ala Leu Leu Ala SerPro Leu Ala Ile Phe Arg Glu Tyr Ser Leu 180 185 190 ATT GAG ATC ATT CCGGAT TTT GAG ATT GTG GCC TGT ACT GAA AAA TGG 683 Ile Glu Ile Ile Pro AspPhe Glu Ile Val Ala Cys Thr Glu Lys Trp 195 200 205 CCT GGC GAG GAA AAGAGC ATC TAT GGC ACT GTC TAC AGT CTT TCT TCC 731 Pro Gly Glu Glu Lys SerIle Tyr Gly Thr Val Tyr Ser Leu Ser Ser 210 215 220 TTG TTG ATC CTG TACGTT TTG CCT CTG GGC ATA ATA TCA TTT TCC TAC 779 Leu Leu Ile Leu Tyr ValLeu Pro Leu Gly Ile Ile Ser Phe Ser Tyr 225 230 235 ACT CGC ATT TGG AGTAAA TTG AAG AGC CAT GTC AGT CCT GGA GCT GCA 827 Thr Arg Ile Trp Ser LysLeu Lys Ser His Val Ser Pro Gly Ala Ala 240 245 250 255 AAT GAC CAC TACCAT CAG CGA AGG CAA AAA ACC ACC AAA ATG CTG GTG 875 Asn Asp His Tyr HisGln Arg Arg Gln Lys Thr Thr Lys Met Leu Val 260 265 270 TGC GTG GTG GTGGTG TTT GCG GTC AGC TGG CTG CCT CTC CAT GCC TTC 923 Cys Val Val Val ValPhe Ala Val Ser Trp Leu Pro Leu His Ala Phe 275 280 285 CAG CTT GCC GTTGAC ATT GAC AGC CAT GTC CTG GAC CTG AAG GAG TAC 971 Gln Leu Ala Val AspIle Asp Ser His Val Leu Asp Leu Lys Glu Tyr 290 295 300 AAA CTC ATC TTCACA GTG TTC CAC ATC ATC GCC ATG TGC TCC ACT TTT 1019 Lys Leu Ile Phe ThrVal Phe His Ile Ile Ala Met Cys Ser Thr Phe 305 310 315 GCC AAT CCC CTTCTC TAT GGC TGG ATG AAC AGC AAC TAT AGA AAG GCT 1067 Ala Asn Pro Leu LeuTyr Gly Trp Met Asn Ser Asn Tyr Arg Lys Ala 320 325 330 335 TTC CTC TCTGCC TTC CGC TGT GAG CAG CGG TTG GAT GCC ATT CAC TCT 1115 Phe Leu Ser AlaPhe Arg Cys Glu Gln Arg Leu Asp Ala Ile His Ser 340 345 350 GAG GTG TCCGTG ACA TTC AAG GCT AAA AAG AAC CTG GAG GTC AGA AAA 1163 Glu Val Ser ValThr Phe Lys Ala Lys Lys Asn Leu Glu Val Arg Lys 355 360 365 AAT AGT GGCCCC AAT GAC TCT TTC ACA GAA GCT ACC AAT GTC 1205 Asn Ser Gly Pro Asn AspSer Phe Thr Glu Ala Thr Asn Val 370 375 380 TAAGGAAGCT AGGGTGTGAAAATGTATGAA TGAATTCTGA CCAGAGCTAT AAATCTGGTT 1265 GATGGCGGCT CACAAGTGATAATTGATTTC CCATTTTAAG GAAGAAGAGG ATCTAAATGG 1325 AAGCATCTGC TGTTTAGTTCCTGGAAAACT GGCTGGGAAG AGCCTGTGTG AAAATACTTG 1385 AATTCAAAGA TAAGGCAGCAAAATGGTTTA CTTAACAGTT GGTAGGGTAG TAGGTTGAAT 1445 TAGGAGTAAA AGCAGAGAGAGGTACTTTTG ACTATTTTCC TGGAGTGAAG TAAACTTGAA 1505 CAAGGAATTG GTATTATCAGCATTGCAAAG AGACGGTGGG TAAATAAGTT GATTTTCAGA 1565 TTTCATTAGG ACCTGGATTGGGGAGCTGTG TAGTTCACGG TTCCCTGCTT GGCTGATGAA 1625 AACGTCGCTG AACAAAAATTTCTCCAGGGA GCCACAGGCT CTCCTTCATC ACGTTTTGAT 1685 TTTTTTTGTT AATTCTCTAGACAAAATCCA TCAAGGAATG CTGCAGGAAA AGATTGCCAG 1745 CTATATGAAT GGCTTCAAGGAACTAAACTG AAACTTGCTA TATAATTAAT ATTTTGGCAG 1805 ACGATAGGGG AACTCCTCAACACTCAGTGA GCCAATTGTT CTTAAAACCG GTTGCACATT 1865 TGGTGAAAGT TTCTTCAACTCTGAATCAAA AGCTGAAATT CTCAGAATTG CAGGAAATGC 1925 AAACCATCAT TTAATTTGTAATTTCAAGTT ACATCTGCTT TATGGAGATA TTTAGATAAC 1985 AAGCATACAA CTTGATAGTTTTATTGTTAT ACCTTTTTGA ACATGTATGA TTTATGTTAT 2045 TATTCCTATT GGAGCTAAGTTTGTCTACAC TAAAATTTAA ATCAGAATAA AGAATAATTT 2105 TTGTGGAAAA AAAAAAAAAAAAAAAAAAAA AAACTCGAG 2144 381 amino acids amino acid linear protein 2Met Gly Pro Ile Gly Thr Glu Ala Asp Glu Asn Gln Thr Val Glu Glu 1 5 1015 Met Lys Val Glu Gln Tyr Gly Pro Gln Thr Thr Pro Arg Gly Glu Leu 20 2530 Val Pro Asp Pro Glu Pro Glu Leu Ile Asp Ser Thr Lys Leu Ile Glu 35 4045 Val Gln Val Val Leu Ile Leu Ala Tyr Cys Ser Ile Ile Leu Leu Gly 50 5560 Val Ile Gly Asn Ser Leu Val Ile His Val Val Ile Lys Phe Lys Ser 65 7075 80 Met Arg Thr Val Thr Asn Phe Phe Ile Ala Asn Leu Ala Val Ala Asp 8590 95 Leu Val Val Asn Thr Leu Cys Leu Pro Phe Thr Leu Thr Tyr Thr Leu100 105 110 Met Gly Glu Trp Lys Met Gly Pro Val Leu Cys His Leu Val ProTyr 115 120 125 Ala Gln Gly Leu Ala Val Gln Val Ser Thr Ile Thr Leu ThrVal Ile 130 135 140 Ala Leu Asp Arg His Arg Cys Ile Val Tyr His Leu GluSer Lys Ile 145 150 155 160 Ser Lys Arg Ile Ser Phe Leu Ile Ile Gly LeuAla Trp Gly Ile Ser 165 170 175 Ala Leu Leu Ala Ser Pro Leu Ala Ile PheArg Glu Tyr Ser Leu Ile 180 185 190 Glu Ile Ile Pro Asp Phe Glu Ile ValAla Cys Thr Glu Lys Trp Pro 195 200 205 Gly Glu Glu Lys Ser Ile Tyr GlyThr Val Tyr Ser Leu Ser Ser Leu 210 215 220 Leu Ile Leu Tyr Val Leu ProLeu Gly Ile Ile Ser Phe Ser Tyr Thr 225 230 235 240 Arg Ile Trp Ser LysLeu Lys Ser His Val Ser Pro Gly Ala Ala Asn 245 250 255 Asp His Tyr HisGln Arg Arg Gln Lys Thr Thr Lys Met Leu Val Cys 260 265 270 Val Val ValVal Phe Ala Val Ser Trp Leu Pro Leu His Ala Phe Gln 275 280 285 Leu AlaVal Asp Ile Asp Ser His Val Leu Asp Leu Lys Glu Tyr Lys 290 295 300 LeuIle Phe Thr Val Phe His Ile Ile Ala Met Cys Ser Thr Phe Ala 305 310 315320 Asn Pro Leu Leu Tyr Gly Trp Met Asn Ser Asn Tyr Arg Lys Ala Phe 325330 335 Leu Ser Ala Phe Arg Cys Glu Gln Arg Leu Asp Ala Ile His Ser Glu340 345 350 Val Ser Val Thr Phe Lys Ala Lys Lys Asn Leu Glu Val Arg LysAsn 355 360 365 Ser Gly Pro Asn Asp Ser Phe Thr Glu Ala Thr Asn Val 370375 380 2144 base pairs nucleic acid single linear mRNA 3 UGAUUGAGGUACAAGUUGUA GACUCUUGUG CUGGUUGCAG GCCAAGUGGA ACUGUACUGA 60 AAAUGGGUCCAAUAGGUACA GAGGCUGAUG AGAACCAGAC AGUGGAAGAA AUGAAGGUGG 120 AACAAUAUGGGCCACAAACC ACUCCUAGAG GUGAACUGGU CCCUGAUCCU GAGCCAGAGC 180 UUAUAGAUAGUACCAAGCUG AUUGAGGUAC AAGUUGUCCU CAUAUUGGCC UAUUGCUCCA 240 UCAUCUUGCUUGGGGUAAUU GGCAACUCCU UGGUGAUCCA CGUGGUGAUC AAAUUCAAGA 300 GCAUGCGCACAGUAACCAAC UUUUUCAUCG CCAAUCUGGC UGUGGCAGAU CUUGUGGUGA 360 AUACUCUGUGUCUACCAUUC ACUCUUACCU ACACCUUAAU GGGGGAGUGG AAAAUGGGUC 420 CUGUCCUGUGCCACCUGGUG CCCUAUGCAC AGGGCCUGGC AGUACAAGUA UCCACAAUCA 480 CCUUGACAGUAAUUGCCCUG GACCGGCACA GGUGCAUCGU CUACCACCUG GAGAGCAAGA 540 UCUCCAAGCGUAUCAGCUUC CUGAUUAUUG GCUUGGCCUG GGGCAUCAGU GCCCUGCUAG 600 CAAGUCCCCUGGCCAUCUUC CGGGAGUAUU CACUGAUUGA GAUCAUUCCG GAUUUUGAGA 660 UUGUGGCCUGUACUGAAAAA UGGCCUGGCG AGGAAAAGAG CAUCUAUGGC ACUGUCUACA 720 GUCUUUCUUCCUUGUUGAUC CUGUACGUUU UGCCUCUGGG CAUAAUAUCA UUUUCCUACA 780 CUCGCAUUUGGAGUAAAUUG AAGAGCCAUG UCAGUCCUGG AGCUGCAAAU GACCACUACC 840 AUCAGCGAAGGCAAAAAACC ACCAAAAUGC UGGUGUGCGU GGUGGUGGUG UUUGCGGUCA 900 GCUGGCUGCCUCUCCAUGCC UUCCAGCUUG CCGUUGACAU UGACAGCCAU GUCCUGGACC 960 UGAAGGAGUACAAACUCAUC UUCACAGUGU UCCACAUCAU CGCCAUGUGC UCCACUUUUG 1020 CCAAUCCCCUUCUCUAUGGC UGGAUGAACA GCAACUAUAG AAAGGCUUUC CUCUCUGCCU 1080 UCCGCUGUGAGCAGCGGUUG GAUGCCAUUC ACUCUGAGGU GUCCGUGACA UUCAAGGCUA 1140 AAAAGAACCUGGAGGUCAGA AAAAAUAGUG GCCCCAAUGA CUCUUUCACA GAAGCUACCA 1200 AUGUCUAAGGAAGCUAGGGU GUGAAAAUGU AUGAAUGAAU UCUGACCAGA GCUAUAAAUC 1260 UGGUUGAUGGCGGCUCACAA GUGAUAAUUG AUUUCCCAUU UUAAGGAAGA AGAGGAUCUA 1320 AAUGGAAGCAUCUGCUGUUU AGUUCCUGGA AAACUGGCUG GGAAGAGCCU GUGUGAAAAU 1380 ACUUGAAUUCAAAGAUAAGG CAGCAAAAUG GUUUACUUAA CAGUUGGUAG GGUAGUAGGU 1440 UGAAUUAGGAGUAAAAGCAG AGAGAGGUAC UUUUGACUAU UUUCCUGGAG UGAAGUAAAC 1500 UUGAACAAGGAAUUGGUAUU AUCAGCAUUG CAAAGAGACG GUGGGUAAAU AAGUUGAUUU 1560 UCAGAUUUCAUUAGGACCUG GAUUGGGGAG CUGUGUAGUU CACGGUUCCC UGCUUGGCUG 1620 AUGAAAACGUCGCUGAACAA AAAUUUCUCC AGGGAGCCAC AGGCUCUCCU UCAUCACGUU 1680 UUGAUUUUUUUUGUUAAUUC UCUAGACAAA AUCCAUCAAG GAAUGCUGCA GGAAAAGAUU 1740 GCCAGCUAUAUGAAUGGCUU CAAGGAACUA AACUGAAACU UGCUAUAUAA UUAAUAUUUU 1800 GGCAGACGAUAGGGGAACUC CUCAACACUC AGUGAGCCAA UUGUUCUUAA AACCGGUUGC 1860 ACAUUUGGUGAAAGUUUCUU CAACUCUGAA UCAAAAGCUG AAAUUCUCAG AAUUGCAGGA 1920 AAUGCAAACCAUCAUUUAAU UUGUAAUUUC AAGUUACAUC UGCUUUAUGG AGAUAUUUAG 1980 AUAACAAGCAUACAACUUGA UAGUUUUAUU GUUAUACCUU UUUGAACAUG UAUGAUUUAU 2040 GUUAUUAUUCCUAUUGGAGC UAAGUUUGUC UACACUAAAA UUUAAAUCAG AAUAAAGAAU 2100 AAUUUUUGUGGAAAAAAAAA AAAAAAAAAA AAAAAAAACU CGAG 2144

We claim:
 1. An isolated nucleic acid encoding a polypeptide whichcomprises the amino acid sequence which is given by SEQ ID NO:2.
 2. Avector comprising an isolated nucleic acid molecule containing asequence encoding polypeptide as claimed in claim 1 where in saidsequence encoding said polypeptide is selected from the group consistingof: (a) nucleotides 63 to 1205 of SEQ ID NO:1; and (b) nucleotides 63 to1205 of SEQ ID NO:3.
 3. A vector as claimed in claim 2 wherein theisolated nucleic acid molecule is deoxyribonucleic acid.
 4. A vector asclaimed in claim 2 which comprises (a).
 5. A vector as claimed in claim2 wherein the isolated nucleic acid molecule is ribonucleic acid.
 6. Anexpression vector which comprises a nucleic acid molecule as claimed inclaim 1 operably linked to regulatory elements necessary for expressionof the nucleic acid molecule.
 7. An expression vector as claimed inclaim 6 for use in a host cell wherein the host cell is Escherichiacoli.
 8. An expression vector as claimed in claim 6 for use in a hostcell wherein the host cell is a mammalian host cell.
 9. An expressionvector as claimed in claim 8 which further comprises the BK virusenhancer.
 10. An expression vector as claimed in claim 9 which furthercomprises an adenovirus late promoter.
 11. A host cell which istransfected with an expression vector as claimed in claim
 6. 12. Atransfected host cell as claimed in claim 11 which is transfectedEscherichia coli.
 13. A transfected host cell as claimed in claim 11which is a transfected mammalian host cell.